Lentivirus-based gene transfer vectors

ABSTRACT

A recombinant lentiviral vector expression system comprises a first vector that comprises a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome. The vector contains at least one defect in at least one gene encoding an EIAV structural protein, but is preferably a gag/pol expression vector. The expression system further comprises a second vector, also comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, and additionally containing a multiple cloning site wherein a heterologous gene may be inserted. The expression system also comprises a third vector which expresses a viral envelope protein. The first and third vectors are packaging signal-defective. When the expression system is transfected into a lentivirus-permissive cell, replication-defective EIAV particles may be produced, which particles are useful in delivering heterologous genes to a broad range of both dividing and non-dividing cells.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication Ser. No. 60/046,891, filed May 13, 1997, which applicationis incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to viruses as vectors useful in genedelivery, and more specifically to lentiviral vectors useful in genedelivery to non-dividing and dividing cells.

BACKGROUND OF THE INVENTION

[0003] The capacities to introduce a particular foreign or native genesequence into a mammalian cell and to control the expression of thatgene are of substantial value in the fields of medical and biologicalresearch. Such capacities provide a means for studying gene regulation,and for designing a therapeutic basis for the treatment of disease.

[0004] The introduction of a particular foreign or native gene into amammalian host cell is facilitated by introducing a gene sequence into asuitable nucleic acid vector. A variety of methods have been developedwhich are capable of permitting the introduction of such a recombinantvector into a desired host cell. In contrast to methods which involveDNA transformation or transfection, the use of viral vectors can resultin the rapid introduction of the recombinant molecule in a wide varietyof host cells. In particular, viral vectors have been employed in orderto increase the efficiency of introducing a recombinant nucleic acidvector into host cells. Viruses that have been employed as vectors forthe transduction and expression of exogenous genes in mammalian cellsinclude SV40 virus (see, e.g. H. Okayama et al., Molec. Cell. Biol. 5,1136-1142 (1985)); bovine papilloma virus (see, e.g., D. DiMaio et al.,Proc. Natl. Acad. Sci. USA 79, 4030-4034 (1982)); adenovirus (see, e.g.,J. E. Morin et al., Proc. Natl. Acad. Sci. USA 84, 4626 (1987)),adeno-associated virus (AAV; see, e.g., N. Muzyczka et al., J. Clin.Invest. 94, 1351 (1994)); herpes simplex virus (see, e.g., A. I. Geller,et al., Science 241, 1667 (1988)), and others.

[0005] Retrovirus-based vectors are particularly favored as tools toachieve stable, integrated gene transfer of foreign genes into mammaliancells. Retroviruses that have been employed as vectors for theintroduction and expression of exogenous genes in mammalian cellsinclude the Moloney murine sarcoma virus (T. Curran et al., J. Virol.44, 674-682 (1982); A. Gazit et al, J. Virol. 60, 19-28 (1986)) andmurine leukemia viruses (MuLV; A. D. Miller, Curr. Top. Microbio.Immunol. 158, 1-24 (1992).

[0006] Efforts to introduce recombinant molecules into mammalian cellshave been hampered by the inability of many cells to be infected by theabove-described viral or retroviral vectors. Limitations on retroviralvectors, for example, include a relatively restricted host range, basedin part on the level of expression of the membrane protein that servesas the viral receptor. M. P. Kavanaugh et al., Proc. Natl. Acad. Sci USA91, 7071-7075 (1994). Other limitations include the inability tointegrate into non-dividing cells (e.g., neurons, hepatocytes,myofibers, hematopoietic stem cells), modest vector titers availablewith current packaging systems, and the fragility of vector particlesthat precludes purification and concentration.

[0007] Lentiviruses are a subgroup of retroviruses that are capable ofinfecting non-dividing cells. L. Naldini et al. report a lentiviralvector system based on the human immunodeficiency virus (HIV) that iscapable of transducing heterologous gene sequences intonon-proliferative HeLa cells and rat fibroblasts, as well as into humanprimary macrophages and terminally differentiated neurons. Science 272,263-267 (1996). However, the use of such a system in humans raisesserious safety concerns, due to the possibility of recombination by thevector into a virulent and disease-causing form.

[0008] Accordingly, a need remains for a safe and efficient lentiviralvector systems capable of mediating gene transfer into a broad range ofdividing and non-dividing cells.

SUMMARY OF THE INVENTION

[0009] The present invention is directed to the transfer of heterologousgene sequences into cells using Equine Infectious Anemia Virus(EIAV)-derived vectors for gene delivery.

[0010] A first aspect of the present invention is a recombinantlentiviral vector expression system including a first vector comprisinga nucleic acid sequence of at least part of the Equine Infectious AnemiaVirus (EIAV) genome, wherein the vector (i) contains at least one defectin at least one gene encoding an EIAV structural protein, and (ii)contains a defective packaging signal. The expression systemadditionally includes a second vector comprising a nucleic acid sequenceof at least part of the EIAV genome, wherein the vector (i) contains acompetent packaging signal, and (ii) contains a multiple cloning sitewherein a heterologous gene may be inserted. The vector expressionsystem also includes a third vector comprising a nucleic acid sequenceof a virus, wherein the third vector (i) expresses a viral envelopeprotein, and (ii) contains a defective packaging signal.

[0011] A second aspect of the present invention is a method of producinga replication-defective lentivirus particle, comprising transfecting acell with a vector expression system of the invention as describedabove.

[0012] A third aspect of the present invention is a method of deliveringa heterologous gene to a target cell, comprising transfecting saidtarget cell with a vector expression system of the invention asdescribed above.

[0013] A fourth aspect of the present invention is a method of producinga lentiviral stock comprising (a) transfecting a producer cell with avector expression system of the invention as described above; (b)growing the producer cell under cell culture conditions sufficient toallow production of replication-defective lentivirus particles in thecell; and (c) collecting the replication-defective lentivirus particlesfrom the producer cell.

[0014] The foregoing and other aspects of the invention are explained indetail in the drawings herein and the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a schematic illustration of plasmid constructs used togenerate EIAV-derived vectors of the present invention. Only a portionof each plasmid is depicted.

[0016]FIG. 2A is a schematic illustration of the plasmid pEV53.

[0017]FIG. 2B is a schematic illustration of the plasmid pEV53A.

[0018]FIG. 3 is a schematic illustration of the plasmid pEC-lacZ.

[0019]FIG. 4 is a schematic illustration of the plasmid pEC-puro.

[0020]FIG. 5 is a schematic illustration of the plasmid pCI-VSV-G.

[0021]FIG. 6A contains four photographs illustrating gene transfer todividing (left column) and non-dividing (right-column) cells by MuLV(top row) and EIAV vectors (bottom row), as described in Example 3,below.

[0022]FIG. 6B is a graphical representation of the comparativeefficiency of gene transfer to dividing and non-dividing cells byMuLV-based vectors (left-hand pair of bar graphs) and EIAV-based vectors(right-hand pair of bar graphs). Successful gene transfer is representedon the y-axis of the graph as the percentage of infected cells that arestained blue (i.e., are X-gal positive), as described below.

[0023]FIG. 6C is a graphical representation of the ability ofaphidicolin to block the incorporation of bromodeoxyuridine (BrdU) intoDNA in cells infected by MuLV-based gene transfer vectors. Successfulgene transfer is represented on the y-axis of the graph as thepercentage of infected cells that are positive for BrdU, as described inExample 3, below.

[0024]FIG. 7 contains two graphs illustrating the ability of theEIAV-based vector EC-lacZ to infect and transfer genes dividing andnon-dividing cells. In the left-hand graph of FIG. 7, open circlesrepresent human CFT1 cells not treated with aphidicolin, while closedcircles represent human CFT1 cells treated with aphidicolin. In theright-hand graph of FIG. 7, open triangles represent equine dermal cellsnot treated with aphidicolin, while closed triangles represent equinedermal cells treated with aphidicolin. In both graphs, gene transfer isrepresented on the y-axis as the percentage of infected cells that areX-gal positive. In both graphs, dosage of the EC-lacZ vector isrepresented on the x-axis in units of μL virus/mL inoculum.

[0025]FIG. 8 is a dose-response curve of the infectivity of eitherunconcentrated (uncentrifuged) or concentrated (centrifuged)EC-lacZ/VSV-G pseudotyped virus particles. In FIG. 8, open circlesrepresent concentrated virus particles, while closed circles representunconcentrated virus particles. Dosage of the EC-lacZ vector isrepresented on the x-axis in units of μL virus/mL inoculum, whileinfectivity is represented on the y-axis as percentage of cells positivefor X-gal.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention now will be described more fullyhereinafter with reference to the accompanying drawings, in whichpreferred embodiments of the invention are shown. This invention may,however, be embodied in many different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will fully convey thescope of the invention to those skilled in the art.

[0027] I. The Equine Infectious Anemia Virus Genome

[0028] The Equine Infectious Anemia Virus (EIAV) is a member of thelentivirus genus of the retrovirus family. The wild type EIAV virus hasa dimeric RNA genome (single-stranded, positive polarity) that ispackaged into a spherical enveloped virion containing a nucleoproteincore. Replication of the wild type EIAV genome occurs via reversetranscription and integration into the host cell genome. The genomecontains three genes that encode the structural proteins gag, pol, andenv, and long terminal repeats (LTR) at each end of the integrated viralgenome. In addition to the gag, pol, and env sequences common to allretroviruses, the EIAV genome contains several short open reading frames(ORFs). These short ORFs are translated from multiply spliced mRNAs. ORFS1 encodes the transcriptional transactivator tat. ORF S2 encodes aprotein whose function is unknown, and the ORF S3 appears to encode arev protein. It is thought that rev is required for the efficientexpression of gag, pol and env. Rev acts post-transcriptionally byinteracting with an RNA sequence known as the rev-responsive element(RRE), which is located in EIAV within the env gene.

[0029] The wild type genome of EIAV also contains several cis-actingsequences, including the R sequence (short repeat at each end of thegenome); the U5 sequence (unique sequence element immediately after theR sequence); the U3 sequence (unique sequence element located downstreamfrom the structural proteins); promoter elements that controltranscriptional initiation of the integrated provirus; a packagingsequence (herein referred to interchangeably as a packaging site or apackaging signal); and a 5′-splice donor site.

[0030] II. EIAV Vectors of the Present Invention

[0031] The vectors of the present invention provide a means forreplicating and expressing heterologous nucleic acid independent of thehost cell nucleus in a broad phylogenetic range of host cells. Thisvector-mediated incorporation of heterologous nucleic acid into a hostcell is referred to as transfection or infection of the host cell,wherein infection means the use of virus particles, and transfectionmeans the use of naked molecules of nucleic acid.

[0032] The vectors of the present invention additionally permit theincorporation of heterologous nucleic acid into virus particles, therebyproviding a means for amplifying the number of infected host cellscontaining heterologous nucleic acid therein. The incorporation of theheterologous nucleic acid facilitates the replication of theheterologous nucleic acid within the viral particle, and the subsequentproduction of a heterologous protein therein. A heterologous protein isherein defined as a protein or fragment thereof wherein all or a portionof the protein is not expressed by the host cell. A nucleic acid or genesequence is said to be heterologous if it is not naturally present inthe wild type of the viral vector used to deliver the gene into a cell(e.g., the wild-type EIAV genome). The term nucleic acid sequence orgene sequence, as used herein, is intended to refer to a nucleic acidmolecule (preferably DNA). Such gene sequences may be derived from avariety of sources including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such gene sequences may comprise genomic DNA whichmay or may not include naturally occurring introns. Moreover, suchgenomic DNA may be obtained in association with promoter sequences orpoly-adenylation sequences. The gene sequences of the present inventionare preferably cDNA. Genomic or cDNA may be obtained in any number ofways. Genomic DNA can be extracted and purified from suitable cells bymeans well-known in the art. Alternatively, mRNA can be isolated from acell and used to prepare cDNA by reverse transcription, or other means.

[0033] It is an object of this invention to generate vectors capable ofcarrying out one single round of replication in the process ofdelivering a gene of interest to a target cell. An aspect of thistechnology is a gene delivery system that excludes the transfer of viralgenes to the target cell. This is accomplished by physically separatingexpression vectors encoding viral genes from the vector encoding thegene of interest. The separate expression vectors are transfected into apermissive cell (e.g., a “producing cell”). Viral gene products arenecessary for the production of virus particles. However, in the presentinvention, genes coding for these genes are located on expressionvectors that contain defective packaging signals; accordingly, the genesthat code for the structural proteins are not packaged.

[0034] The phrases “structural protein” or “EIAV structural protein” asused herein refer to the encoded proteins which are required forencapsidation (e.g., packaging) of the EIAV genome, and include gag, poland env.

[0035] The term “defective” as used herein refers to a nucleic acidsequence that is not functional with regard to either encoding its geneproduct or serving as a signaling sequence. To illustrate, a defectiveenv gene sequence will not encode the env protein; a defective packagingsignal will not facilitate the packaging of the nucleic acid moleculethe defective signal is located on. Nucleic acid sequences may be madedefective by any means known in the art, including by the deletion ofsome or all of the sequence, by placing the sequence out-of-frame, or byotherwise blocking the sequence.

[0036] As used herein, the terms “deleted” or “deletion” mean eithertotal deletion of the specified segment or the deletion of a sufficientportion of the specified segment to render the segment inoperative ornonfunctional, in accordance with standard usage. The term “replicationdefective” as used herein, means that the vectors that encode EIAVstructural proteins cannot be encapsidated in the target cell aftertransfection of the vectors. The resulting lentivirus particles arereplication defective inasmuch as the packaged vector does not includeall of the viral structural proteins required for encapsidation, atleast one of the required structural proteins being deleted therefrom,such that the packaged vector is not capable of replicating the entireviral genome.

[0037] The preferred vectors of the present invention are derived fromEIAV. Native EIAV nucleic acid may be isolated from cells infected withthe virus, and vectors prepared therefrom. An exemplary method forpreparing EIAV vectors is provided in S. T. Perry, et al., J. Virol. 66,4085-4097 (1992). For example, cDNA may be produced from EIAV RNA byreverse transcriptase, using methods known in the art. Double-strandedEIAV cDNA may then be produced and cloned into a cloning vector, such asa bacterial cloning vector. Any cloning vector, such as bacterial, yeastor eukaryotic vectors, known and used by those skilled in the art, maybe used. The vectors of the present invention preferably comprise cDNAcomplementary to at least part of the RNA genome of EIAV. One vector maycontain an heterologous cDNA molecule, which molecule can be introducedinto human or animal cells to achieve transcription or expression of theheterologous molecule. The cDNA molecules will comprise cDNAcomplementary to at least part of a EIAV genome and comprising part ofRNA genome required for replication of the genome, with the cDNAmolecule being placed under transcriptional control of a promotersequence functional in the cell.

[0038] A promoter sequence of the present invention may comprise apromoter of eukaryotic or prokaryotic origin, and will be sufficient todirect the transcription of a distally located sequence (i.e. a sequencelinked to the 5′ end of the promoter sequence) in a cell. The promoterregion may also include control elements for the enhancement orrepression of transcription. Suitable promoters are the cytomegalovirusimmediate early promoter (pCMV), the Rous Sarcoma virus long terminalrepeat promoter (pRSV), and the SP6, T3, or T7 promoters. Enhancersequences upstream from the promoter or terminator sequences downstreamof the coding region may be optionally be included in the vectors of thepresent invention to facilitate expression. Vectors of the presentinvention may also contain additional nucleic acid sequences, such as apolyadenylation sequence, a localization sequence, or a signal sequence,sufficient to permit a cell to efficiently and effectively process theprotein expressed by the nucleic acid of the vector. Exampled ofpreferred polyadenylation sequences are the SV40 early regionpolyadenylation site (C. V. Hall et al., J. Molec. App. Genet. 2, 101(1983)) and the SV40 late region polyadenylation site (S. Carswell andJ. C. Alwine, Mol. Cell Biol. 9, 4248 (1989)). Such additional sequencesare inserted into the vector such that they are operably linked with thepromoter sequence, if transcription is desired, or additionally with theinitiation and processing sequence if translation and processing aredesired. Alternatively, the inserted sequences may be placed at anyposition in the vector. The term “operably linked” is used to describe alinkage between a gene sequence and a promoter or other regulatory orprocessing sequence such that the transcription of the gene sequence isdirected by an operably linked promoter sequence, the translation of thegene sequence is directed by an operably linked translational regulatorysequence, and the post-translational processing of the gene sequence isdirected by an operably linked processing sequence.

[0039] Standard techniques for the construction of the vectors of thepresent invention are well-known to those of ordinary skill in the artand can be found in such references as Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989). Avariety of strategies are available for ligating fragments of DNA, thechoice of which depends on the nature of the termini of the DNAfragments and which choices can be readily made by the skilled artisan.

[0040] In one embodiment of the present invention, a recombinantlentiviral expression system comprises three vectors. The first vectorcomprises a nucleic acid sequence of at least part of the EquineInfectious Anemia Virus (EIAV) genome, wherein the vector (i) containsat least one defect in at least one gene encoding an EIAV structuralprotein, and (ii) contains a defective packaging signal. The secondvector comprises a nucleic acid sequence of at least part of the EIAVgenome, wherein the vector (i) contains a competent packaging signal,and (ii) contains a multiple cloning site wherein a heterologous genemay be inserted. The third vector comprises a nucleic acid sequence of avirus, wherein the vector (i) expresses a viral envelope protein, and(ii) contains a defective packaging signal.

[0041] In one embodiment of the invention, the first vector is a gag/polexpression vector. Gag/pol expression vectors express EIAV proteinsrequired for assembly and release of viral particles from cells, andinclude the genes encoding proteins gag and pol. The first vector mayalso express genes encoding the accessory proteins rev and tat. The openreading frame S2, encoding a protein whose function is unknown, mayadditionally be included in the first vector. The first vector isconstructed to contain mutations that exclude retroviral-mediatedtransfer of viral genes. Such mutations may be a deletion of sequencesin the viral env gene, thus excluding the possibility of generatingreplication-competent EIAV, or may be deletions of certain cis-actingsequence elements at the 3′ end of the genome required for viral reversetranscription and integration. Accordingly, even if viral genes fromthis construct are packaged into viral particles, they will not bereplicated and replication-competent wild-type viruses will not begenerated.

[0042] In a preferred embodiment of the invention, the first vector ofthe expression system is the plasmid pEV53, shown in FIG. 2A. pEV53 is a12170 base-pair (bp) plasmid and cDNA clone which at base pairs 209-1072contains a chimeric CMV/EIAV enhancer promoter region located upstreamfrom the EIAV tat coding regions (bp 1124-1210 and 5886-6026), the gagcoding region (bp 1216-2676), the pol coding region (bp 2433-5874) andthe ORF S2 coding region (bp 6037-6234). The vector also contains apartial env coding region (bp 6063-7733) and rev coding regions (bp6188-6288 and 7250-7654). The bovine growth hormone (BGH)polyadenylation signal is provided (bp 7759-7973), as is a phage f1region (bp 8037-8450), an SV40 early promoter region and origin ofreplication (bp 8514-8839), a neomycin resistance coding region (bp9685-9924), a SV40 polyadenylation signal (bp 9685-9924), a Col E1origin of replication (bp 10356-11029), and a β-lactamase (ampicillinresistance) coding region (bp 11174-12035).

[0043] In a more preferred embodiment of the present invention, thefirst vector of the expression system is the plasmid pEV53A, shown inFIG. 2B. The plasmid pEV53A is derived from the pEV53 plasmid, whereinmodifications have been made to further reduce the chances ofretroviral-mediated transfer of viral genes without affecting theexpression levels of EIAV proteins. In the plasmid pEV53A, all EIAV longterminal repeat (LTR) sequences containing promoter/enhancer elementsand cis-acting sequence elements important for integration and the tRNAprimer binding site sequence for initiation of reverse transcription hasbeen deleted from pEV53. pEV53A is constructed from pEV53 by deletingnucleotides 902 through 1077 of pEV53.

[0044] The second vector of the expression system of the presentinvention is designed to serve as the vector for gene transfer, andcontains all cis-acting sequence elements required to support reversetranscription (replication) of the vector genome, as well as a multiplecloning site for insertion of cDNAs encoding heterologous genes ofinterest. In the present invention, the vector encoding the gene ofinterest is a recombinant EIAV-derived vector that carries the geneticinformation to be transduced into a target cell, along with cis-actingsequence elements necessary for the packaging and integration of theviral genome. The second vector will preferably contain some portion ofthe gag coding sequence, as it is believed that certain parts of the gagsequence play a role in the packaging of the EIAV genome. Moreover, the5′ splice donor site contained in the LTR will preferably contain amutation that increases the titer of the produced virus, as describedin, e.g. W. Tan et al., J. Virol 70, 3645-3658 (1996).

[0045] Two examples of preferred second vectors are provided in FIGS. 3and 4. FIG. 3 illustrates the plasmid and cDNA clone pEC-lacZ, a 8749 bpplasmid derived from EIAV that expresses the E. Coli lacZ reporter gene.The plasmid contains two CMV immediate-early enhancer promoter regions(located at bp 1-734 and 1609-2224), R-U5 sequence domains from the EIAVlong terminal repeat (LTR) (bp 735-849), a partial EIAV gag sequence (bp993-1570), the lacZ coding sequence (bp 2297-5437), a EIAV LTR sequence(bp 5752-6073), a phage f1 region (bp 6163-6618), an ampicillinresistance coding region (bp 7057-7917) and a ColE1 origin ofreplication (bp 8062-8736).

[0046]FIG. 4 illustrates the plasmid and cDNA clone pEC-puro, a 6099 bpplasmid derived from EIAV expressing the puromycin resistance gene. Thisvector also contains two CMV immediate-early enhancer promoter regions(bp 1-734 and 1609-2224), R-U5 sequence domains from the EIAV longterminal repeat (LTR) (bp 735-849), a partial gag sequence from EIAV (bp993-1570), the puromycin resistance gene coding sequence (bp 2334-2933),an EIAV LTR sequence (bp 3102-3423), a phage f1 region (bp 3513-3968),an ampicillin resistance coding region (bp 4407-5267) and a ColE1 originof DNA replication (bp 5412-6086).

[0047] As will be appreciated by one skilled in the art, the nucleotidesequence of the inserted heterologous gene sequence or sequences may beof any nucleotide sequence. For example, the inserted heterologous genesequence may be a reporter gene sequence or a selectable marker genesequence. A reporter gene sequence, as used herein, is any gene sequencewhich, when expressed, results in the production of a protein whosepresence or activity can be monitored. Examples of suitable reportergenes include the gene for galactokinase, beta-galactosidase,chloramphenicol acetyltransferase, beta-lactamase, etc. Alternatively,the reporter gene sequence may be any gene sequence whose expressionproduces a gene product which affects cell physiology. Heterologous genesequences of the present invention may comprise one or more genesequences that already possess on ore more promoters, initiationsequences, or processing sequences.

[0048] A selectable marker gene sequence is any gene sequence capable ofexpressing a protein whose presence permits one to selectively propagatea cell which contains it. Examples of selectable marker genes includegene sequences capable of conferring host resistance to antibiotics(e.g., puromycin, ampicillin, tetracycline, kanamycin, and the like), orof conferring host resistance to amino acid analogues, or of permittingthe growth of bacteria on additional carbon sources or under otherwiseimpermissible culture conditions. A gene sequence may be both a reportergene and a selectable marker gene sequence. The most preferred reportergenes of the present invention are the lacZ gene which encodes thebeta-galactosidase activity of E. Coli; and the gene encoding puromycinresistance.

[0049] Preferred reporter or selectable marker gene sequences aresufficient to permit the recognition or selection of the vector innormal cells. In one embodiment of the invention, the reporter genesequence will encode an enzyme or other protein which is normally absentfrom mammalian cells, and whose presence can, therefore, definitivelyestablish the presence of the vector in such a cell.

[0050] The heterologous gene sequence may also comprise the codingsequence of a desired product such as a suitable biologically activeprotein or polypeptide, immunogenic or antigenic protein or polypeptide,or a therapeutically active protein or polypeptide. Alternatively, theheterologous gene sequence may comprise a sequence complementary to anRNA sequence, such as an antisense RNA sequence, which antisensesequence can be administered to an individual to inhibit expression of acomplementary polynucleotide in the cells of the individual.

[0051] Expression of the heterologous gene may provide immunogenic orantigenic protein or polypeptide to achieve an antibody response, whichantibodies can be collected from an animal in a body fluid such asblood, serum or ascites.

[0052] In a preferred embodiment of the present invention, the thirdvector of the recombinant lentiviral expression system expresses a viralenvelope protein. Such a vector will accordingly comprise a nucleic acidsequence encoding a viral protein under the control of a suitablepromoter. It is possible to alter the host range of cells that the viralvectors of the present invention can infect by utilizing an envelopegene from another closely related virus. In other words, it is possibleto expand the host range of the EIAV vectors of the present invention bytaking advantage of the capacity of the envelope proteins of certainviruses to participate in the encapsidation of other viruses. In aparticularly preferred embodiment of the present invention, theG-protein of vesicular-stomatitis virus (VSV-G; see, e.g., Rose andGillione, J. Virol. 39, 519-528 (1981); Rose and Bergmann, Cell 30,753-762 (1982)), or a fragment or derivative thereof, is the envelopeprotein expressed by the third vector. VSV-G efficiently formspseudotyped virions with genome and matrix components of other viruses.As used herein, the term “pseudotype” refers to a viral particle thatcontains nucleic acid of one virus but the envelope protein of anothervirus. In general, VSV-G pseudotyped vectors have a very broad hostrange, and may be pelleted to titers of high concentration byultracentrifugation (e.g., according to the method of J. C. Burns, etal., Proc. Natl. Acad. Sci. USA 90, 8033-8037 (1993)), while stillretaining high levels of infectivity.

[0053] An illustrative and preferred example of a third vector of thepresent invention is shown in FIG. 5. This Figure illustrates theplasmid and cDNA clone pCI-VSV-G, a preferred expression vector for theenvelope glycoprotein VSV-G. The plasmid contains 5679 base pairs andincludes the CMV immediate-early enhancer promoter region (bp 1-795), achimeric intron region (bp 857-989), the VSV-G coding region (bp1088-2633), a phage f1 region (3093-3548), a SV40 late polyadenylationsignal (bp 2782-3003), a ColE1 origin of DNA replication (bp 4992-5666)and an ampicillin resistance coding region (bp 3987-4847).

[0054] In a method of the present invention, infectious,replication-defective EIAV particles may be prepared according to themethods disclosed herein in combination with techniques known to thoseskilled in the art. The method includes transfecting anlentivirus-permissive cell with the vector expression system of thepresent invention; producing the EIAV-derived particles in thetransfected cell; and collecting the virus particles from the cell. Thestep of transfecting the lentivirus-permissive cell can be carried outaccording to any suitable means known to those skilled in the art. Forexample, in a method of the present invention, the three-plasmidexpression system described herein is used to generate EIAV-derivedretroviral vector particles by transient transfection. As anotherexample, uptake of the vectors into the cells can be achieved by anysuitable means, such as for example, by treating the cells withDEAE-dextran, treating the RNA with “LIPOFECTIN®” before addition to thecells, or by electroporation. These techniques are well known in theart.

[0055] The step of facilitating the production of the infectious viralparticles in the cells may also be carried out using conventionaltechniques, such as by standard cell culture growth techniques.

[0056] The step of collecting the infectious virus particles may also becarried out using conventional techniques. For example, the infectiousparticles may be collected by cell lysis, or collection of thesupernatant of the cell culture, as is known in the art. Optionally, thecollected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

[0057] If desired by the skilled artisan, lentiviral stock solutions maybe prepared using the vectors and methods of the present invention.Methods of preparing viral stock solutions are known in the art and areillustrated by, e.g., Y. Soneoka et al., Nucl. Acids Res. 23, 628-633(1995) and N. R. Landau et al., J Virol. 66, 5110-5113 (1992). In amethod of producing a stock solution in the present invention,lentiviral-permissive cells (referred to herein as producer cells) aretransfected with the vector system of the present invention. The cellsare then grown under suitable cell culture conditions, and thelentiviral particles collected from either the cells themselves or fromthe cell media as described above. Suitable producer cell lines include,but are not limited to, the human embryonic kidney cell line 293, theequine dermis cell line NBL-6, and the canine fetal thymus cell lineCf2TH.

[0058] The vectors of the present invention are also useful in preparingstable packaging cells (i.e. cells that stably express EIAV virusproteins, which cells, by themselves, cannot generate infectious virusparticles). Methods for preparing packaging cells that expressretrovirus proteins are known in the art and are exemplified by themethods set forth in, for example, U.S. Pat. No. 4,650,764 to Temin etal., which disclosure is incorporated herein in its entirety. Within thescope of the present invention, a packaging cell will comprise alentivirus-permissive host cell comprising an EIAV nucleic acid sequencecoding for at least one EIAV structural protein, which nucleic acidsequence is packaging-signal defective, thus rendering the cell itselfcapable of producing at least one EIAV structural protein, but notcapable of producing replication-competent infectious virus. In oneembodiment, the EIAV nucleic acid sequence is an EIAV gag-pol expressionvector such as, for example, pEV53. A packaging cell may be made bytransfecting an EIAV-permissive host cell (e.g., a human embryonickidney 293 cell) with a suitable EIAV nucleic acid sequence as providedabove according to known procedures. The resulting packaging cell isthus able to express and produce at least one EIAV structural protein.However, in that the EIAV nucleic acid sequence is defective in thepackaging signal, the cell, on its own, is not able to producereplication-competent EIAV virus. The packaging cell may then betransfected with other nucleic acid sequences (e.g., pEC-puro, pEC-lacZor pCI-VSV-G), which may contain heterologous genes of interest and anappropriate packaging signal. Once transfected with the additionalsequence or sequences, the packaging cell may thus be used to providestocks of EIAV viruses that contain heterologous genes, but whichviruses are themselves replication-incompetent.

[0059] Pharmaceutical formulations, such as vaccines, of the presentinvention comprise an immunogenic amount of the infectious, replicationdefective virus particles as disclosed herein in combination with apharmaceutically acceptable carrier. An “immunogenic amount” is anamount of the infectious virus particles which is sufficient to evoke animmune response in the subject to which the pharmaceutical formulationis administered. An amount of from about 10³ to about 10⁷ virusparticles, and preferably about 10⁴ to 10⁶ virus particles per dose isbelieved suitable, depending upon the age and species of the subjectbeing treated, and the immunogen against which the immune response isdesired. Exemplary pharmaceutically acceptable carriers include, but arenot limited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution. Subjects which may be administeredimmunogenic amounts of the infectious, replication-defective virusparticles of the present invention include but are not limited to humanand animal (e.g., pig, cattle, dog, horse, donkey, mouse, hamster,monkeys) subjects.

[0060] Pharmaceutical formulations of the present invention includethose suitable for parenteral (e.g., subcutaneous, intradermal,intramuscular, intravenous and intraarticular), oral or inhalationadministration. Alternatively, pharmaceutical formulations of thepresent invention may be suitable for administration to the mucusmembranes of a subject (e.g., intranasal administration). Theformulations may be conveniently prepared in unit dosage form and may beprepared by any of the methods well known in the art.

[0061] The vectors and methods of the present invention are useful in invitro expression systems, wherein the inserted heterologous geneslocated on the second vector encode proteins or peptides which aredesirably produced in vitro.

[0062] The vectors, methods and pharmaceutical formulations of thepresent invention are additionally useful in a method of administering aprotein or peptide to a subject in need of the desired protein orpeptide, as a method of treatment or otherwise. In this embodiment ofthe invention, the heterologous gene located on the second vector of thepresent invention encodes the desired protein or peptide, and helpercells or pharmaceutical formulations containing the helper cells of thepresent invention are administered to a subject in need of the desiredprotein or peptide. In this manner, the protein or peptide may thus beproduced in vivo in the subject. The subject may be in need of theprotein or peptide because the subject has a deficiency of the proteinor peptide, or because the production of the protein or peptide in thesubject may impart some therapeutic effect, as a method of treatment orotherwise, and as explained further below.

[0063] III. Gene Transfer Technology

[0064] The gene transfer technology of the present invention has severalapplications. The most immediate applications are perhaps in elucidatingthe process of peptides and functional domains of proteins. Cloned cDNAor genomic sequences for proteins can be introduced into different celltypes in culture, or in vivo, in order to study cell-specificdifferences in processing and cellular fate. By placing the codingsequences under the control of a strong promoter, a substantial amountof the desired protein can be made. Furthermore, the specific residuesinvolved in protein processing, intracellular sorting, or biologicalactivity can be determined by mutational change in discrete residues ofthe coding sequences.

[0065] Gene transfer technology of the present invention can also beapplied to provide a means to control expression of a protein and toassess its capacity to modulate cellular events. Some functions ofproteins, such as their role in differentiation, may be studied intissue culture, whereas others will require reintroduction into in vivosystems at different times in development in order to monitor changes inrelevant properties.

[0066] Gene transfer provides a means to study the nucleic acidsequences and cellular factors which regulate expression of specificgenes. One approach to such a study would be to fuse the regulatoryelements to be studied to reported genes and subsequently assaying theexpression of the reporter gene.

[0067] Gene transfer also possesses substantial potential use inunderstanding and providing therapy for disease states. There are anumber of inherited diseases in which defective genes are known and havebeen cloned. In some cases, the function of these cloned genes is known.In general, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, at least sometimes involving regulatoryor structural proteins, which are inherited in a dominant manner. Fordeficiency state diseases, gene transfer could be used to bring a normalgene into affected tissues for replacement therapy, as well as to createanimal models for the disease using antisense mutations. For unbalanceddisease states, gene transfer could be used to create a disease state ina model system, which could then be used in efforts to counteract thedisease state. Thus the methods of the present invention permit thetreatment of genetic diseases. As used herein, a disease state istreated by partially or wholly remedying the deficiency or imbalancewhich causes the disease or makes it more severe. The use ofsite-specific integration of nucleic sequences to cause mutations or tocorrect defects is also possible.

[0068] Hematopoietic stem cells, lymphocytes, vascular endothelialcells, respiratory epithelial cells, keratinocytes, skeletal and musclecardiac cells, neurons and cancer cells are among proposed targets fortherapeutic gene transfer, either ex vivo or in vivo. See, e.g., A. D.Miller, Nature 357, 455-460 (1992); R. C. Mulligan, Science 260, 926-932(1993). These cells and others are suitable target cells for the vectorsand methods of the present invention.

[0069] In summary, the viral vectors of the present invention can beused to stably transfect either dividing or non-dividing cells, andstably express a heterologous gene. Using this vector system, it is nowpossible to introduce into dividing or non-dividing cells, genes whichencode proteins that can affect the physiology of the cells. The vectorsof the present invention can thus be useful in gene therapy for diseasestates, or for experimental modification of cell physiology.

[0070] Having now described the invention, the same will be illustratedwith reference to certain examples which are included herein for thepurposes of illustration only, and which are not intended to be limitingof the invention.

EXAMPLE 1 Plasmid Construction

[0071] The parent plasmid for EIAV vectors described herein is theplasmid pER2.1, generously provided by Dr. Fred Fuller of North CarolinaState University, Raleigh, N.C., USA. The construction of the clonepER2.1 is described in S. T. Perry et al., J. Virol. 66, 4085-4097(1992). pER2.1 encodes an infectious DNA clone of the Malmquist Wyomingstrain of EIAV. One safety aspect of this clone is that the virusgenerated does not cause disease in its natural equine host. Also,unlike vectors derived from other retroviruses (e.g., murine leukemiavirus, human immunodeficiency virus) considered for use in genetransfer, wild type EIAV does not mount an active infection in humancells.

[0072] EIAV gag/pol (first) expression vector. The pEV53 plasmid (shownin FIG. 2A) was designed to express EIAV proteins required for assemblyand release of viral particles form cells, and includes genes encodingproteins encoded by the gag and pol genes, and the accessory proteinsrev and tat. The open reading frame S2, encoding a protein whosefunction is not known, was also included. pEV53 was constructed tocontain mutations that exclude retroviral-mediated transfer of viralgenes. The mutations include sequence deletions in the viral env gene(thus excluding the possibility of generating replication-competent EIAVvirus particles) and deletion of certain cis-acting sequence elements atthe 3′ end of the genome required for viral reverse transcription andintegration. Deletions are included such that in the unlikely event thatviral genes are packaged into the infectious particles, they will not bereplicated (e.g., replication-competent wild-type vectors will not begenerated). The plasmid pEV53A, shown in FIG. 2B, is derived from pEV53,but differs from pEV53 in that all EIAV long terminal repeat (LTR)sequences containing promoter/enhancer elements and cis-acting sequenceelements important for integration, as well as the tRNA primer bindingsite sequence for initiation of reverse transcription, have been deletedby removing nucleotides 902 through 1077 from pEV53). The pEV53A plasmidwas made to further reduce the chances of retroviral-mediated transferof viral genes without affecting the expression levels of EIAV proteins

[0073] Gene Transfer (second) vector. The pEC-lacZ (shown in FIG. 3) andpEC-puro (shown in FIG. 4) plasmids were designed to serve as the vectorfor gene transfer and contain all cis-acting sequence elements requiredto support reverse transcription (e.g., replication) of the vectorgenome, as well as a multiple cloning site for insertion of cDNAsencoding genes of interest. The pEC-lacZ plasmid encodes thebeta-galactosidase reporter gene lacZ, while the pEC-puro plasmidencodes the puromycin-N-acetyl transferase dominant selectable markergene puro.

[0074] Viral envelope gene expression (third) vector. The third plasmidof the expression system described herein is the plasmid pCI-VSV-G,shown in FIG. 5. This plasmid expresses a viral envelope gene,specifically the vesicular stomatitis virus G glycoprotein gene.

EXAMPLE 2 Production and Testing of Viral Vectors

[0075] EIAV vectors were produced following standard calciumphosphate-mediated co-transfection of the pEV53A gag-pol expressionplasmid, the pCI-VSV-G env expression plasmid, and either the pEC-lacZor pEC-puro expression vector plasmids into cultures of human 293 cells(American Type Culture Collection, Rockville, Md.). 48 hours aftertransfection, the culture medium was harvested from the cells and testedfor EIAV vector production. The gene transfer efficiency of the pEC-purovector was measured by the ability of serial dilutions of the vector toconfer resistance to the drug puromycin. In this assay, an infected cellgives rise to a drug-resistant colony of cells, which can then becounted. Human 293 cells were used as the target cells. From sixindependent experiments, the average titer of the EC-puro vector wasdetermined to be 2±1×10⁵ colony forming units (cfu) per mL.

[0076] The gene transfer efficiency of the EC-lacZ vector was determinedby staining infected human CFT1 human airway epithelial cells with X-gal(Molecular Probes, Inc., Eugene, Oreg.), an analog of galactose. In thisassay, cells expressing the beta-galactosidase gene will turn blue, andthe percentage of blue cells is determined by counting. The titer ofvirus by multiplying the fraction of stained cells by the number ofcells initially infected. In this manner, the titer of the EC-lacZ viruswas determined to be about 5±1×10⁴ infectious units (n=5) per mL.

[0077] Several control experiments were performed to determine whetherthe expression of the puro and lacZ genes were indeed mediated by viralcomponents. In the first experiment, it was shown for both the EC-puroand EC-lacZ vectors that both the pEV53 or pEV53A and the pCI-VSV-Gvectors were essential for gene transfer. The result is consistent withgene transfer and expression mediated by virus (and not free DNA). In asecond control experiment using the lacZ vector, cells were stained withX-gal immediately after a two-hour infection and no blue cells werefound. The result is consistent with temporal aspects of reversetranscription, integration and gene expression, which for otherretroviruses are known to require a minimum of about 10 hours tocomplete.

EXAMPLE 3 Gene Transfer to Non-Dividing Cells

[0078] EIAV is capable of infecting non-dividing, terminallydifferentiated cells, such macrophages and airway epithelia. To testthis property with the present invention, human CFT1 airway epithelialcells (generously provided by J. R. Yankaskas of the University of NorthCarolina at Chapel Hill) were arrested in the cell cycle withaphidicolin (Calbiochem-Novabiochem Corp., La Jolla, Calif.), and theninfected with the pEC-lacZ vector. As a control, cells were infected inparallel with the LZ vector, a lacZ-containing retrovirus vector derivedfrom the murine leukemia virus (MuLV). 48 hours after infection,cultures were stained for beta-galactosidase activity with X-gal. In theaphidicolin treated cultures, aphidicolin was present both during andafter infection.

[0079] Microscopic fields of the stained cells are shown in FIG. 6A. TheLZ vector efficiently infected cells not treated with aphidicolin (upperleft panel). However, when cells were arrested in the cell cycle byaphidicolin treatment, gene transfer efficiency dropped markedly (upperright panel). It was estimated by counting the rare blue-stained cellthat the relative efficiency of gene transfer to dividing cells tonon-dividing cells was about 100 to 1 for the LZ vector (as shown inFIG. 6B, left-hand pair of bar graphs). The results are consistent withresults obtained in experiments in other laboratories. See e.g., D. G.Miller et al., Mol. Cell. Biol. 10, 4329-4242 (1990); P. F. Lewis and M.Emerman, J. Virol., 68, 510-516 (1994). At the time of infection,parallel cultures were pulsed with bromodeoxyuridine (BrdU) for 2 hoursto test the efficacy of the aphidicolin block and analyzed for BrdUincorporation into DNA, as shown in FIG. 6C. It was found thataphidicolin markedly reduced the incorporation of BrdU into DNA.

[0080] In contrast to the results obtained with the LZ vector,aphidicolin treatment had no significant effect on the percentage ofhuman CFT1 cells infected by the EC-lacZ vector (see results in FIGS.6A, lower panels, and 6B, right-hand pair of bar graphs). These resultsindicate that EIAV vectors can efficiently infect non-dividing cells.For example, in the experiment shown in FIG. 7, the equine dermalfibroblast cell line NBL-6 was infected about 10 times more readily (fora given amount of virus), when cells were treated with aphidicolin, ascompared to cells that were not treated with aphidicolin.

EXAMPLE 4 Ultracentrifugation of VSV-G Pseudotyped EIAV Vectors

[0081] One advantage of VSV-G pseudotyped vectors is that the increasedstability afforded by VSV-G permits concentration of infectivity bypelleting in an ultracentrifuge (see, e.g., J. C. Burns, et al., Proc.Natl. Acad. Sci. USA 90, 8033-8037 (1993)). It was determined thatinfectivity of VSV-G pseudotyped EIAV vectors can also be recovered bypelleting using centrifugation techniques. In this experiment, 720 ml ofEC-lacZ-containing supernatant from a 2-plasmid (pEC-lacZ/pCI-VSV-G)co-transfection of 293 cells (stably modified to express the pEV53plasmid) was concentrated by pelleting the virus in a high-speedcentrifuge. The pellet was suspended in 0.36 ml of 1×Hank's BalancedSalt Solution (HBSS, Cat. # 14175, Life Technologies, Inc.,Gaithersburg, Md.) to achieve a 2000-fold concentration of virusparticles. The infectivity was determined (see FIG. 8 for adose-response curve of infectivity) and it was found that the titerincreased about 1200-fold from 5×10⁵ to 6×10⁸. Thus, the yield ofinfectivity was about 60%. This result illustrates that EIAV vectors canbe concentrated to high titers by pelleting.

EXAMPLE 5 Packaging Cell Line

[0082] For stable packaging cells, the human embryonic kidney cell line293 was transfected using a standard calcium transfection procedure withpEV53. The cells were selected for expression of neomycin using G418(geneticin) (Life Technologies Inc., Gaithersburg Md., USA). Individualcolonies were selected, expanded and tested for virus production aftertransfecting with pECpuro and pCI-VSV-G. Clonal packaging cell linesshowing the greatest vector production were either frozen for long-termstorage or were maintained in culture for analysis of persistence ofpackaging function. It was found that the packaging function was stablefor at least one month, indicating that the present invention is usefuland successful in the preparation of stable, EIAV-based packaging celllines.

[0083] While the invention has been described in connection withspecific embodiments thereof, it will be understood that the inventionis capable of further modification and this application is intended tocover any variations, uses or adaptations of invention following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains, and as may be applied tothe essential features set forth in the scope of the appended claims.

That which is claimed:
 1. A recombinant lentiviral vector expressionsystem comprising: (a) a first vector comprising a nucleic acid sequenceof at least part of the Equine Infectious Anemia Virus (EIAV) genome,wherein said vector (i) contains at least one defect in at least onegene encoding an EIAV structural protein, and (ii) contains a defectivepackaging signal; (b) a second vector comprising a nucleic acid sequenceof at least part of the EIAV genome, wherein said vector (i) contains acompetent packaging signal, and (ii) contains a multiple cloning sitewherein a heterologous gene may be inserted; and (c) a third vectorcomprising a nucleic acid sequence of a virus, wherein said third vector(i) expresses a viral envelope protein, and (ii) contains a defectivepackaging signal.
 2. A vector system according to claim 1 , wherein saidsecond vector is deficient for expression of at least one EIAVstructural protein.
 3. A vector system according to claim 1 , whereinsaid first vector, said second vector, and said third vector are cDNAclones of at least part of the EIAV genome.
 4. A vector expressionsystem according to claim 1 , wherein said first vector is a gag-polexpression vector, and wherein said vector contains a defect in the envgene.
 5. A vector expression system according to claim 4 , wherein saiddefect in the env gene is a deletion mutation.
 6. A vector expressionsystem according to claim 1 , wherein said first vector and said secondvector each contain a defect in the env gene.
 7. A vector expressionsystem according to claim 1 , wherein said third vector encodes anenvelope protein that is not an EIAV envelope protein.
 8. A vectorexpression system according to claim 1 , wherein said third vectorexpresses the vesicular stomatitis virus G glycoprotein.
 9. A vectorexpression system according to claim 1 , wherein said second vectorcontains a heterologous gene.
 10. A vector expression system accordingto claim 9 , wherein said heterologous gene encodes an antigenic proteinor peptide.
 11. A vector expression system according to claim 1 ,wherein said first vector is selected from the group consisting of theplasmid pEV53 and the plasmid pEV53A; said second vector is selectedfrom the group consisting of pEC-lacZ and pEC-puro; and said thirdvector is the plasmid pCI-VSV-G.
 12. The plasmid set forth in FIG. 2A aspEV53.
 13. The plasmid set forth in FIG. 2B as pEV53A.
 14. The plasmidset forth in FIG. 2 as pEC lacZ.
 15. The plasmid set forth in FIG. 3 aspEC-puro.
 16. The plasmid set forth in FIG. 4 as pCI-VSV-G.
 17. A methodof producing a replication-defective lentivirus particle, comprisingtransfecting a cell with: (a) a first vector comprising a nucleic acidsequence of at least part of the Equine Infectious Anemia Virus (EIAV)genome, wherein said vector (i) contains at least one defect in at leastone gene encoding an EIAV structural protein, and (ii) contains adefective packaging signal; (b) a second vector comprising a nucleicacid sequence of at least part of the EIAV genome, wherein said vector(i) contains a competent packaging signal, and (ii) contains a multiplecloning site wherein a heterologous gene may be inserted; and (c) athird vector comprising a nucleic acid sequence of a virus, wherein saidthird vector (i) expresses a viral envelope protein, and (ii) contains adefective packaging signal.
 18. A method according to claim 17 , whereinsaid cell is a non-dividing cell.
 19. A method according to claim 17 ,wherein said second vector contains a heterologous gene.
 20. Areplication-defective lentivirus particle produced according to themethod of claim 17 .
 21. An infectious EIAV particle containing an EIAVnucleic acid sequence encoding a promoter and a heterologous genesequence, and wherein said nucleic acid sequence is defective inencoding at least one EIAV structural protein so that said virusparticle is replication defective.
 22. A cell containing areplication-defective lentiviral particle, wherein said lentiviralparticle is produced according to the method of claim 17 .
 23. A methodof delivering a heterologous gene to a target cell, comprisingtransfecting said target cell with: (a) a first vector comprising anucleic acid sequence of at least part of the Equine Infectious AnemiaVirus (EIAV) genome, wherein said vector (i) contains at least onedefect in at least one gene encoding an EIAV structural protein, and(ii) contains a defective packaging signal; (b) a second vectorcomprising a nucleic acid sequence of at least part of the EIAV genome,wherein said vector (i) contains a competent packaging signal, and (ii)contains a multiple cloning site wherein a heterologous gene may beinserted; and (c) a third vector comprising a nucleic acid sequence of avirus, wherein said third vector (i) expresses a viral envelope protein,and (ii) contains a defective packaging signal.
 24. A method accordingto claim 23 , wherein said target cell is a non-dividing cell.
 25. Amethod of producing a lentiviral stock comprising: (a) transfecting alentivirus-permissive producer cell with (i) a first vector comprising anucleic acid sequence of at least part of the Equine Infectious AnemiaVirus (EIAV) genome, wherein said vector (1) contains at least onedefect in at least one gene encoding an EIAV structural protein, and (2)contains a defective packaging signal; (ii) a second vector comprising anucleic acid sequence of at least part of the EIAV genome, wherein saidvector (1) contains a competent packaging signal, (2) contains aheterologous gene; and (iii) a third vector comprising a nucleic acidsequence of a virus, wherein said third vector (1) expresses a viralenvelope protein, and (2) contains a defective packaging signal; (b)growing said producer cell under cell culture conditions sufficient toallow production of replication-defective lentivirus particles in saidcell; and (c) collecting said replication-defective lentivirus particlesfrom said producer cell.
 26. A method according to claim 25 , whereinsaid producer cell is grown in a cell culture medium, and wherein saidreplication-defective lentivirus particles are collected from saidmedium.
 27. A method of delivering a heterologous gene to a target cell,comprising infecting said target cell with replication-defectivelentivirus particles collected according to step (c) of claim 25 .
 28. Amethod according to claim 27 , wherein said target cell is anon-dividing cell.
 29. A method according to claim 27 , wherein saidtarget cell is a human airway epithelial cell.
 30. A method ofdelivering a heterologous gene to a target cell, comprising infectingsaid target cell with a replication-defective lentivirus particleaccording to claim 20 or 21 .
 31. A pharmaceutical formulationcomprising a replication-defective lentivirus particle according toclaim 20 or claim 21 in a pharmaceutically acceptable carrier.
 32. Amethod of making a packaging cell, comprising transfecting alentivirus-permissive cell with a vector comprising a nucleic acidsequence of at least part of the Equine Infectious Anemia Virus (EIAV)genome, wherein said vector contains a defective packaging signal.
 33. Amethod according to claim 32 , wherein said vector is a gal-polexpression vector.
 34. A method according to claim 32 , wherein saidvector is selected from the group consisting of the plasmid pEV53 andthe plasmid pEV53A.
 35. A method according to claim 32 , wherein saidlentivirus-permissive cell is a human 293 cell.
 36. A packaging cellcomprising a lentivirus-permissive host cell containing an EIAV nucleicacid sequence encoding at least one EIAV structural protein, whereinsaid nucleic acid sequence is packaging-signal defective, such that thecell itself is capable of producing at least one EIAV structuralprotein, but not capable of producing replication-competent infectiousvirus.